Lyme borreliosis is a chronic multisystem disorder caused by the tick- borne bacterium Borrelia burgdorferi. In light of the facts that approximately 10% of cases of primary Lyme borreliosis escape clinical detection and that untreated disease may have debilitating long term consequences, there is a need to develop a Lyme borreliosis vaccine. Attachment of invasive microorganisms to host cells is a critical early step in disease pathogenesis. Previous investigators have demonstrated that B. burgdorferi attaches to mammalian cells in vitro, but the mechanisms of attachment have not been elucidated. Preliminary studies performed by the PI indicate that virulent B. burgdorferi possess a moderately high affinity glycosaminoglycan binding ligand which is putatively surface exposed. It is hypothesized that B. burgdorferi attach to host cell-surface exposed proteoglycans. This proposal aims to characterize glycosaminoglycan-mediated borrelial attachment to mammalian cells at the molecular level. The effects of glycosaminoglycans on borrelial attachment to mammalian cells will be characterized in detail in attachment-inhibition studies using a variety of cell types and a number of virulent and avirulent B. burgdorferi strains (Specific aim #1). The B. burgdorferi glycosaminoglycan-binding ligand will be purified by heparin agarose affinity chromatography and will be subjected to N-terminal and internal amino acid sequence analysis (Specific aim #2). Monoclonal antibodies will be generated against the protein and will be assessed for their ability to inhibit borrelial attachment (Specific aim #2). The glycosaminoglycan binding ligand will be cloned, sequenced, and expressed in Escherichia coli (Specific aim #3). Recombinant B. burgdorferi glycosaminoglycan binding ligand will be purified by a combination of heparin agarose affinity chromatography and immunoaffinity chromatography; it will be assessed in in vitro attachment-inhibition studies and detailed studies will be undertaken to define the epitope recognized by antibodies which inhibit attachment (Specific aim #4). In the future, it is planned to characterize the glycosaminoglycan binding domain of the protein and to assess the purified recombinant protein as a vaccinogen in the murine model of Lyme borreliosis (future directions).